Masked (or “caged”) fluorescent dyes initially exist in a non-fluorescent form, which may be transformed into the fluorescent state (“uncaged”) by an external stimulus, i.e. enzyme, light, change of the pH-value, etc. This process my be reversible or irreversible, but in most cases “uncaging” means an irreversible change in the course of which the initially colourless or only slightly yellowish substance turns to be strongly coloured and fluorescent. Light is a treasured stimulus in life sciences for several reasons. It may be applied non-invasively, at various wavelengths, with various powers, and with a very high spatial and temporal precision. Therefore, photochemically induced uncaging processes comprise an important tool-box in life sciences and biophysical chemistry. The initially invisible “caged” fluorescent dye may be randomly distributed in the studied object, or it may be used as a label, e.g. in the form of a bioconjugate which “recognizes” the target. After that, a certain dose of UV or visible light produces the coloured and fluorescent species, so that their spatial and temporal distribution may be controlled with high precision at the moment of “uncaging” and later, as the system evolves. Such “optical injections” may generate fluorescent marks not only in biological systems, but also in capillars of the microfluidic sytems [P. H. Paul, M. G. Garguilo, D. J. Rakestraw, Anal. Chem. 1998, 70, 2459-2467; J. I. Molho, A. E. Herr, B. P. Mosier, J. G. Santiago, T. W. Kenny, R. A. Brennen, G. Gordon, B. Mohammadi, Anal. Chem. 2001, 73, 1350-1360]. Imaging of the newly created fluorescent mark followed by its monitoring in time provides quantitative dynamic and structural parameters. For choosing fluorophores for caging, several parameters are important: uncaging efficiency under standard illumination conditions (diode lasers or lamps at wavelengths >375 nm), quick photoactivation (in the range of milliseconds or faster) for tracking rapid processes, high fluorescence quantum yields and photostability after uncaging, ability to penetrate into living cells (alone and after binding with “small” molecules which recognize target structures), low (photo)toxicity of the caged substances and their (decomposition) products after uncaging, high performance in water and aqueous buffers, reliable synthesis and conjugation protocols, stability of bioconjugates against hydrolysis, availability of the multicolor “tool-box” with well separated absorption and emission bands for the (co)localization and FRET studies [cf.: R. C. Willis, Anal. Chem. 2007, 79, 1785-1788].
Coumarines [W.-H. Li, YuRui Zhao, U.S. Pat. No. 7,304,168 (Apr. 12, 2007); YuRui Zhao, Q. Zheng, K. Dakin, K. Xu, M. Martinez, W.-H. Li, J. Am. Chem. Soc. 2004, 126, 4653-4663], fluorescein [a) R. P. Haugland, K. R. Gee, U.S. Pat. No. 5,635,608 (Mar. 6, 1997); b) T. Kobayashi, Y. Urano, M. Kamiya, T. Ueno, H. Kojima, T. Nagano, J. Am. Chem. Soc. 2007, 129, 6696-6697; c) G. A. Krafft, W. R. Sutton, R. T. Cummings, J. Am. Chem. Soc. 1988, 110, 301-303] and rhodamines [a) Rhodamine Q: T. J. Mitchison, K. E. Slavin, J. A. Theriot, K. Gee, A. Mallavarapu, Methods in Enzymology 1998, 291, 63-78; Bioorg. Med. Chem. Lett. 2001, 11, 2181-2183; b) Rhodamine 110: L. D. Lavis, T.-Y. Chao, R. T. Raines, ACS Chem. Biol. 2006, 1, 252; J. Ottl, D. Gabriel, G. Marriott, Bioconjugate Chem. 1998, 9, 143-151] have been disclosed and used as caged fluorescent dyes. Synthesis and properties of caged fluorescent 2-amidothioxanthones—compounds structurally similar to fluorescein or rhodamines—were also reported [J. R. R. Majjigapu, A. N. Kurchan, R. Kattani, T. P. Gustafson, A. G. Kutateladze, J. Am. Chem. Soc. 2005, 127, 12458-12459]. As a photocleavable unit, most of these caged compounds contain a 2-nitrobenzyl group or its derivatives with an α-substituent and/or one or two methoxy groups in the aromatic ring [U.S. Pat. No. 5,635,608]. The α-position to the phenyl ring (CH2— group) may be decorated with a carboxy or an alkyl group facilitating the uncaging reaction. An example of the caged Rhodamine Q is given below. This model compound (compound 1) was synthesized by the present inventors as indicated in Scheme 1.

Methoxy groups in combination with a nitro group in the aromatic ring provide the required absorption in the near UV region and also speed-up the liberation of an uncaged fluorescent dye. Without them the removal of 2-nitrobenzyl group is very slow and requires hard UV light incompatible with living cells. Acylation of both nitrogen atoms in rhodamine Q with commercially available compound 2 affords the yellow compound 1 with a Spiro junction of the xanthene fragment and the aromatic lactone ring. In this non-fluorescent compound the extended π-conjugation is broken, and therefore the deep red colour of rhodamine Q disappeared. Practical applications (e.g. bioconjugation) require compounds with a free second carboxy group or other functionality in the disubstituted “lower” benzene ring. However, the synthesis of all caged rhodamines with such a second carboxy group is difficult and low-yielding [T. J. Mitchison, K. E. Slavin, J. A. Theriot, K. Gee, A. Mallavarapu, Methods in Enzymology 1998, 291, 63-78]. Probably because of that they did not find any widespread use.
A big disadvantage of 2-nitrobenzyl group and its substitutes is that upon photolysis they produce colored and highly reactive 2-nitrosobenzaldehyde or 2-nitrosobenzophenone. All aromatic nitroso compounds including 2-nitrosobenzaldehyde, 2-nitrosobenzophenone and their derivatives are known to be toxic. Acute toxicity of these compounds may unfavourably influence the normal cell life, and even cause cell death. The chemical reactivity of 2-nitrosobenzaldehyde or 2-nitrosobenzophenone is very high; they react with amino groups in proteins giving Schiff's bases (imines) which form oligomers, especially under irradiation with UV-light. The dark colour of these oligomeric by-products may interfere with optical measurements; they may act as a sort of undesired optical filter. The amount of these by-products and oligomers could be reduced, if it were possible to use the monoacylated rhodamine derivatives. Unfortunately, they are still fluorescent (though they absorb and emit at shorter wavelengths than the free dye). An urgent need for other caging groups free from the drawbacks mentioned above stimulates the on-going research. However, other modern caging groups which absorb in the near UV region are also bulky, and the procedures of their synthesis, caging and uncaging reactions may be rather complicated. For example, N,N-dimethyl-2-hydroxy-4-nitrophenol was reported to give photocleavable phenyl esters [A. Banerjee, C. Grewer, L. Ramakrishnan, J. Jäger, A. Gameiro, H.-G. A. Breitinger, K. R. Gee, B. K. Carpenter, G. P. Hess, J. Org. Chem. 2003, 68, 8361-8367; cf.: M. Matsuzaki, G. C. R. Ellis-Davies, T. Nemoto, Y. Miyashita, Y. Iino, H. Kasai, Nature Neurosci. 2001, 4, 1086-1092]. Another heterocycle-7-diethylamino-4-(hydroxymethyl)-2H-chromen-2-one—is known to form esters which may easily be cleaved by irradiation at 412 nm [P. Stegmaier, J. M. Alonso, A. Del Campo, Langmuir 2008, 24, 11872-11879]. Yet other heterocycles—derivatives of 8-bromo-7-hydroxyquinoline [O. D. Fedoryak, T. M. Dore, Org. Lett. 2002, 4, 3419-3422] and 6-bromo-7-hydroxycoumarines [T. Furuta, S. S.-H. Wang, J. L. Dantzker, T. M. Dore, W. J. Bybee, E. M. Callaway, W. Denk, R. Y. Tsien, Proc. Natl. Acad. Sci. USA 1999, 96, 1193-2000; H. Ando, T. Furuta, R. Y. Tsien, H. Okamoto, Nat. Genet. 2001, 28, 317-325; W. Lin, D. S. Lawrence, J. Org. Chem. 2002, 67, 2723-2726; H. J. Montgomery, B. Perdicakis, D. Fishlock, G. A. Lajoie, E. Jervis, J. G. Guilemette, Bioorg. Med. Chem. 2002, 10, 1919-1927]—have also been proposed as photocleavable protecting groups. All of these groups provide the required absorption in the near UV or violet spectral region and may be cleaved off by photoirradiation. The general disadvantage of all bulky caging groups is that their big size and limited solubility in water considerably reduce the cell permeability of the whole assemblies with fluorescent dyes. Further conjugation with small molecules is sometimes required for selective binding with the biological targets. These additional structural fragments further increase the molecular dimensions and may retard or even inhibit the penetration of the whole photoactivable adducts through cell walls and bio-membranes precluding their successful use in biological microscopy.
Consequently, the main object of the present invention was to provide novel photoactivable fluorescent dyes incorporating small photoactive groups with improved properties such as the capability for easy and effective photoactivation (uncaging) which does not generate toxic substances which may interfere with biochemical processes in cells or tissues.
A related object was to provide novel reagents and methods for biocojugation and various imaging techniques, including those which provide optical single molecule switching (SMS) “nanoscopy” (diffraction unlimited optical resolution by using switching of the fluorescence of the single molecules).
These objectives have been achieved by providing the novel photoactivable compounds according to claims 1-3, the methods of preparation according to claims 4-5 and the uses of claims 6-19.